3 protocols
Rockhopper version 1.20 (manuscript submitted) was used for alignment, normalization, and quantification. Rockhopper is available for download from http://cs.wellesley.edu/~btjaden/Rockhopper Genome_build: MGAS5005 genome (NC_007297.1) Supplementary_files_format_and_content: Tab delimited text files containing transcription coordinates and abundance estimates.
nucleic acid library construction protocol
Cultures and two volumes of RNAprotect (Qiagen) added upon reaching the correct growth phase and, following a 5 min room temperature incubation, centrifuged at 4,000g for 10 mins. Bacterial cell pellets were lysed via mechanical lysis (FastPrep machine) and RNA isolated using the miRNeasy kit (Qiagen). Isolated RNA was DNase-treated using TURBO-DNA free (Ambion). rRNAs were removed from the RNA samples using either the RiboZero kit (Epicenter) or TEX. The rRNA-depleted RNAs were used in conjuncation with the ScriptSeq RNA-seq library prep kit (Epicenter). Barcodes were added to allow the four libaries to be multiplexed on a single lane of the illumina instrument. Barcode primers were purchased from Epicenter.
MGAS2221 was grown in Todd-Hewitt broth with 0.2% yeast extract to mid-exponential phase (corresponding to an O.D.600 of 0.5) and to stationary phase (3h after reaching an O.D.600 of 0.5)